Method for identifying members of combinatorial libraries

ABSTRACT

A method to determine the molecular weights of femtomole or smaller quantities of small molecules, such as peptides, oligonucleotides, or heterocyclics, covalently attached to polystyrene beads on a grid, is presented using imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS). The determination is made possible by selectively clipping the bond linking the small molecule to the bead, followed directly by a TOF-SIMS assay of the bead on the grid. The method can be applied to large numbers of polystyrene beads having different small molecules attached thereto for direct characterization of massive combinatorial libraries.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part application of copending U.S. patentapplication Ser. No. 08/217,046, filed Mar. 23, 1994, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a method for the identification andanalysis of members of combinatorial libraries, wherein the identifiedmember has a demonstrated pharmacological or physiological activity.

BACKGROUND OF THE INVENTION

Over the past ten years, there has been a growing demand for theproduction and identification of small molecules that havepharmacological activity as, for example, agonists or antagonists ofvarious cellular acceptor molecules, such as cell-surface receptors,enzymes, or antibodies. Such small molecules can be peptides,oligonucleotides, or other organic compounds, such as heterocyclics andthe like. The unifying feature of these small molecules is operationalin that they bind specifically to known acceptors. In consequence ofsuch binding, a physiological response occurs whereby certain biologicalprocesses are modulated, which can have applications in medicine andagriculture.

Searching for small molecules that are useful as pharmaceuticals entails(1) generating collections of such molecules, (2) screening suchmolecules for physiological activity, and (3) identifying the structureof molecules that provide a positive result in the screen. The first twosteps can be accomplished using methods well-known in the art, some ofwhich are described herein for purposes of clarity. The third step,where one determines the structure of a positively screened smallmolecule, has proven to be the time-limiting step in the overall processto discover new small molecule pharmaceuticals. This step is necessaryto eliminate false positives or duplicates, and, of most importance, toproduce the selected small molecule for a prospective pharmaceuticalformulation.

Searching for such small molecules has involved screening collections ofnatural materials, such as fermentation products, plant or animal tissueextracts, or libraries of synthesized molecules. Chemical assays havebeen designed that merely identify those species that bind a particularacceptor molecule or, in a bioassay, assess the ability of testedmolecules to effect certain physiological reactions. Screening of suchcollections often, at most, provides leads that must be refined by morestringent techniques and expanded testing of related molecules. All ofthese techniques are limited severely by the available concentration ofany particular small molecule and the resolving power of the screeningand analysis techniques. As a result, the process of production andidentification of small molecules that have pharmacological activity, aprocess termed "irrational drug design" by Brenner and Lerner (Proc.Natl. Acad. Sci. USA, 89, 5381-5383 (1992)), "requires continualimprovement both in the generation of repertoires of small molecules!and in the methods of selection." Id. at page 5381.

A repertoire of small molecules, wherein each molecule thereof can berepresented preferably in at least femtomole quantities, typically isproduced by what are termed multiple methods of synthesis or parallelchemical synthetic protocols. Such repertoires are commonly referred toas "combinatorial libraries," for reasons that will become plain below.With reference to peptides, such synthetic methods have been disclosedby Jung and Beck-Sickinger (Angew. Chem. Int. Ed. Engl., 31, 367-383(1992)). Methods for the production of heterocyclic libraries (see Buninand Ellman, J. Am. Chem. Soc., 114, 10997-10998 (1992)) and nucleic acidlibraries (referred to in Brenner and Lerner, supra) have also beenpublished. Other methods for the construction of combinatorial librariesinclude those of Kerr et al., J. Am. Chem. Soc., 115, 2529 (1993); Lamet al., Nature, 354, 82 (1991); Houghten et al., Nature, 354, 84 (1991);and Fodor et al., Science, 251, 767-773 (1991) (see, also U.S. Pat. No.5,143,854 (1992)).

In the methods cited above, members of a library are constructed fromthe coupling of chemical building blocks, such as amino acids, nucleicacids, or variant organic monomers and side groups. Resultant librariesconsist of different individual species, the potential number (k) ofwhich can be calculated as a function of the number of differentbuilding blocks used (a) and the number of different building blockscoupled to each member of the library (b), according to the followingformula: k=a^(b). Thus, a library of pentapeptides constructed using 20different amino acids (i.e., the chemical building blocks) could includeas many as 20⁵ or 3.2 million different species.

The method of Lam et al., supra, is presented as an example of one suchmethod that provides a means to at least approach the theoreticalmaximum number of different species in a combinatorial library. The Lamet al. method employs a "split synthesis" protocol, in which standardsolid phase peptide synthesis (see. e.g., Atherton and Sheppard, SolidPhase Peptide Synthesis, A Practical Approach (Oxford University Press,1989)) is conducted on resin beads. Separate reactions for each aminoacid used take place to couple covalently one amino acid to an aliquotof resin beads. For example, 20 different reaction vessels may be used,in which the resin beads are coupled to one of the 20 naturallyoccurring proteinogenic amino acids. Typically, the amino acids used insuch reactions have been modified using suitable blocking groups knownin the art to allow the coupling of only one amino acid per bead. Aftera first reaction, the aliquots of resin beads having attached theretodifferent single amino acids are combined, thus completing the firstround. A second round to create dipeptides begins by removing theblocking group from the last amino acid added, re-allocating aliquots ofthe resin beads into another 20 reaction vessels, and allowing therebythe coupling of a second single amino acid to each resin bead. Thecombining of the resin beads having dipeptides completes round two. Therounds are repeated until the library of peptides has attained thedesired number of building blocks, which, in this case, are amino acids.

According to the Lam et al. reference, each resin bead processed asrecited above contained about 50 to 200 picomoles of peptide, whichpresumably each consisted of five amino acids. The library can then bescreened for those beads that include peptides that are recognized by aparticular acceptor molecule that is labelled directly or indirectlywith fluorescein or an enzyme, for example, using materials and methodsthat are well known in the art. Such a labelled bead may be isolatedphysically using micromanipulation techniques, or its location, i.e.,address, may be noted for further analysis in situ, i.e., in the midstof the nonselected, unlabelled beads of the library. An alternateapproach, proposed by Brenner and Lerner, supra, would include an"appended `genetic` tag" that would be interpreted to provide thestructure of each molecular species in a library; however, this approachrequires that the genetic tag be added chemically to the individualmolecular species, which could interfere with the ability of a molecularspecies to interact with the acceptor molecule of interest. Even if thegenetic tag presented no such obstacle, such tagged molecular speciesalso would have to be "read" in the midst of multiples of thenon-selected species. The current methods, in essence, have not overcomeadequately the challenge presented in either isolating a labelledmicroscopic bead in view of the large numbers that require analysis(discussed further below) or in readily analyzing the identity of amolecular species attached to a labelled bead when surrounded byidentical, unlabelled beads having different molecular species attachedto them.

Presuming that the bead of interest can be isolated physically, thecontained peptide may be analyzed for its sequence of amino acids usinga commercially available peptide microsequencer, such as Model 477A ofApplied Biosystems, for example. According to Lam et al., although " a!library containing several million beads could be screened with labelledacceptor molecules! in 10-15 Petri dishes in an afternoon , only about!. . . three pentapeptide beads were sequenced daily using themicrosequencer." Evidently, as understood from the technical literaturepresented hereinabove, the limiting step in the process of identifyingnew drugs from combinatorial libraries is the step of discarding falsepositives and determining the identity of the species of interest, whichdifficulty includes the step of either isolating the labelled bead(s)from unlabelled beads or having a sufficiently discerning technologyavailable that can analyze the molecular species on a microscopic beadwhen adjacent to identical beads having different molecular speciesattached thereto.

In the instance of identifying a peptide of interest, for example, thetime limiting step of extracting the sequence of those binding peptides,however, is also limitative in that only peptides containing naturallyoccurring amino acids can be identified. This limitation is due tocharacteristics of the Edman degradation technology upon whichmicrosequencers are based. In addition to having the capability tosequence only a few peptides per day, microsequencers can only sequencepeptides that include naturally-occurring proteinogenic amino acids.

Accordingly, the analysis of any combinatorial library is necessarilyimpeded by the very low rate at which, in the Lam et al.-type method,beads having members of the library attached thereto can be analyzed forthe identity of the attached molecule. In view of the literally millionsof candidate molecules to be screened in a given library, it is probablethat at least hundreds, if not thousands, of the molecularspecies-attached beads would generate positive signals (including falsepositive signals) requiring further analysis. The limitation of beingable to sequence only a few molecules per day, therefore, presents astrong drawback to current strategies of screening combinatoriallibraries for pharmaceutical compounds. Moreover, if a method allowedanalysis of a positively signalled bead having a small molecule ofinterest attached thereto without having to remove such a bead from thegroup of other beads, in the presence of which the bead was screened,the procedure of screening and identifying small molecules of interestwould be greatly improved.

SUMMARY OF THE INVENTION

It has now been discovered that a direct mass spectrometric assay can beconfigured to read a wide variety of combinatorial libraries includingthose composed of peptides, oligonucleotides, and heterocyclicmolecules. Using the present invention, any combinatorial library can beconstructed on a suitable substrate and screened, and the individualsubstrate that is identified as having a molecule that specificallyinteracts with an acceptor molecule of interest (i.e., positive screenresult) can be identified in the presence of identical substrates havingother unselected molecular species attached thereto and subjected todirect mass spectrometric assay without removal from the total libraryto determine the precise molecular weight of the selected molecule. Apreferred aspect of the method includes the use of novel linkingmoieties or substrates having reactive groups attached thereto thatcovalently or ionically link the individual molecules of thecombinatorial library to the substrate, whereby the linkage may bebroken without disturbing the molecule's structure, yet allow thelibrary molecules to remain in close association with the substratebased on physical effects. Consequently, the present invention greatlyimproves the ability of artisans of the relevant art to identifypharmaceutically active agents derived from combinatorial libraries.

Accordingly, the present invention relates to a method of identifyingindividual small molecules of a combinatorial library comprising (a)forming a plurality of complexes of solid substrates and the smallmolecules, each of which comprises one substrate, or portion thereof,and one of the small molecules of said library; and (b) determining themolecular weight of a selected small molecules by means of secondary ionmass spectrometry. Suitable linking moieties and substrates havingsuitable reactive groups attached thereto that connect the smallmolecules to the substrate are disclosed as well.

These and other features and advantages of the invention will be morereadily apparent upon reading the following detailed description of theinvention and upon reference to the accompanying drawings, all of whichare given by way of illustration only, and are not limitative of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the imaging time-of-flight secondaryion mass spectrometry (TOF-SIMS) apparatus.

FIGS. 2(A)-2(C) are a composite of three related mass spectrum profilesof phenylalanine attached to a polystyrene bead by various means, asfollows: by physical adsorption (FIG. 2A); by covalent bonding (FIG.2B); and by physical adsorption after vapor phase clipping withtrifluoroacetic acid (TFA) of linking covalent bond(s) (FIG. 2C).

FIG. 3 is a profile of a tripeptide associated with a polystyrene beadby physical adsorption only and placed on a copper grid.

FIGS. 4(A) and 4(B) are a composite of two TOF-SIMS images, eachdirected at the same address on a copper grid. FIGS. 4A and 4B displaythe (M+H)⁺ ion intensity for phenylalanine and copper, respectively.

FIGS. 5(A) and 5(B) are a composite of two TOF-SIMS images, eachdirected at the same address on a copper grid. FIGS. 5A and 5B displaythe (M+H)⁺ ion intensity for leucine and phenylalanine, respectively.

FIGS. 6(A) and 6(B) are a profile of a tripeptide that was covalentlylinked to a polystyrene bead using an acid vapor labile linkage and thenexposed to acid vapor, and then placed on a copper grid. Included withFIG. 6 is a representation of the structure of the tripeptide V-Y-Vmarked to indicate the fragments identified in the TOF-SIMS profile.

FIGS. 7(A)-7(E) depict the linking moieties attaching the angiotensin IIreceptor antagonist to various polystyrene beads.

FIGS. 8(A) and 8(B) are the composite of two electrospray mass spectraof the angiotensin II receptor antagonist. This data was provided forcomparison with the present invention by S. A. Carr, M. E. Hemling, G.D. Roberts, and J. Weinstock of the Chemical and Biological ResearchDivision of SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.FIG. 8A displays the standard electrospray mass spectrum and FIG. 8Bdisplays the electrospray MS/MS spectrum.

FIGS. 9(A) and 9(B) are the composite of matrix assisted laserdesorption (MALDI) spectra of angiotensin II receptor antagonist. Thisdata was provided for comparison with the present invention by S. A.Carr, M. E. Hemling, G. D. Roberts, and J. Weinstock of the Chemical andBiological Research Division of SmithKline Beecham Pharmaceuticals, Kingof Prussia, Pa. FIG. 9A displays the standard MALDI spectrum and FIG. 9Bdisplays the post source decay spectrum.

FIGS. 10(A)-10(D) are the TOF-SIMS mass spectrum of angiotensin IIreceptor antagonist on a Sasrin bead after cleavage by TFA/CH₂ Cl₂vapors.

FIGS. 11(A) and 11(B) are the composite of two images of the angiotensinII receptor antagonist. FIG. 11A displays the image of the (M+H)⁺ ion(m/z 453.2) and FIG. 11B displays the image of the fragment ion (m/z135).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following detailed description of the preferred embodiments of theinstant invention is provided to aid those skilled in the art inpracticing the present invention, but should not be construed to limitthe present invention, as modifications and variations in theembodiments herein discussed may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

The present invention provides a method and novel materials used in theinventive method that greatly improves the ability of an ordinaryartisan to identify and characterize pharmaceutically-active smallmolecules selected from a combinatorial library. The members of such alibrary preferably are constructed in association with a suitablesubstrate, such as a polystyrene bead surface. Such association betweenthe small molecules and the substrates may be mediated by any suitablemeans, including, but not limited to, physical adsorption, covalentlinkage, ionic bond, hydrophobic interactions, and van der Waals forces.Preferably, such associations are mediated by covalent or ionic linkageduring the construction of such combinatorial libraries, wherein such acovalent or ionic linkage may be broken using means that does not modifyor substantially modify the structure of the linked small molecule, andwherein the small molecule remains in association with the substrate viaphysical adsorption or other effects, but will allow desorption in asecondary ion mass spectrometry (SIMS) apparatus. Construction of such alibrary was described above in the Background section, using the methodof Lam et al. (supra), as an example. Screening of such a library wasalso described above in the Background section. The present inventionrelates to the identification of a positively screened small moleculederived from the aforementioned combinatorial library.

A preferred identification approach would take into account the factthat small molecules, such as peptides, oligonucleotides, orheterocyclic compounds, may be constructed such that they can bedesorbed intact or substantially intact from a substrate, particularlyfrom a bead surface. Because each bead, for example, may have adsorbedthereto only a femtomole quantity of a particular molecular species, orless, extreme sensitivity of the method of analysis is required. Forexample, a standard 40 micron sphere covered with one layer ofphenylalanine will only have about 50 femtomoles of surface moleculesavailable for sampling.

The present method directly assesses the molecular weight of suchmolecular species upon their removal from the substrate and immediatesubsequent ionization. The method employs imaging secondary ion massspectrometry to identify the molecular weights of molecules adsorbed tothe polystyrene bead surfaces, such as magnetic sector SIMS, quadrupoleSIMS, Fourier Transformation SIMS, or time-of-flight SIMS (TOF-SIMS).The methodology actually used for any given SIMS analysis is known inthe art, and may vary both with the machine used and artisan operatingthe machine. Preferably, the present invention employs TOF-SIMS.Detection of the mass of secondary ions formed in a TOF-SIMS protocolallows the unique identification of the corresponding library member,presuming that the method of construction of the library is known sothat an artisan can assign discrete molecular weights to all molecularspecies and ionization fragments thereof (generated in the TOF-SIMSmethod).

In TOF-SIMS, a pulsed beam of primary ions is directed to a samplesurface. The arriving primary ions desorb and ionize molecules of thesample present in a monolayer at the surface of the sample. Thesegenerated secondary ions are then accelerated to a uniform energy by anelectric field, and drift through a fixed distance to a detector. Thetime-of-flight of these uniform energy particles through the fixeddistance is directly proportional to the charge-to-mass ratio (m/z) ofthe ion. Because only the time-of-flight of an ion is measured todetermine its mass, TOF-SIMS provides for parallel detection of allmasses present in a sample, and an effectively unlimited mass detectionrange with high mass resolution. Indeed, TOF-SIMS provides a 10⁴ -10⁶fold improvement in sensitivity over scanning mass spectrometric methodsemploying other detectors, such as magnetic sector fields andquadrapoles, which are well known in the art. TOF-SIMS thus provides adirect mass spectrometric assay that is generally applicable to readinga wide variety of molecular species assembled in combinatoriallibraries.

The considerations relevant to use of TOF-SIMS for such assays arediscussed in the literature. For example, as discussed by Winograd inIon Beams and Laser Postionization for Molecule-Specific Imaging (Anal.Chem., 65, 622A-629A (1993)), an energetic primary ion bombarding asample on a solid surface creates a large amount of damage within 50Angstroms of the point of impact. Unless the dose of incident ions iskept below approximately 1% of the number of sample molecules forming amonolayer, the ion bombardment alters the surface chemistry. The dose ofincident ions of 1% is referred to as the "static limit." In TOF-SIMS,the dosage of primary ions remains below the static limit because theincident ion beam is directed toward the sample as a very short pulse.Use of a pulsed incident beam is also advantageous because a spectrumwith a dynamic range of several orders of magnitude can be obtained bythe accumulation of a large number of cycles with high repetition rates,as discussed by Benninghoven et al. in Surface MS: Probing Real-WorldSamples (Anal. Chem., 65, 630A-639A (1993)). Increased sensitivity mayalso be realized using special cationization schemes or by laserpostionization of sputtered neutral molecules, as discussed by Winogradet al., Inst. Phys. Conf. Ser., 28, 259 (1992).

The TOF-SIMS technique also allows the primary ion beam to be focused toa spot size of less than 150 nm, thereby allowing the concentration ofmolecules to be mapped over small spatial domains by rastoring the ionbeam across pixels defined on the sample and taking spectra at eachpixel. Other aspects of TOF-SIMS imaging are discussed by Chait andStanding in Time-of-Flight Mass Spectrometer for Measurement ofSecondary Ion Mass Spectra (Int. J. Mass Spectrom. Ion Phys., 40,185-193 (1981)); and by Steffens et al. in A Time-of-Flight MassSpectrometer for Static SIMS Applications (J. Vac. Sci. Technol., A3(3), 1322 (1985)).

In certain situations, the information obtained by TOF-SIMS may notfully distinguish and identify all members of a combinatorial library.For example, various isomers of a given peptide may be present, eachhaving the same mass, as, for example, in the case ofphenylalanine-glycine-leucine and glycine-leucine-phenylalanine. In suchsituations, TOF-SIMS can be used to determine the sequence of theselected peptide nonetheless, provided that the library was constructedfrom a known set of building blocks. As discussed by Poppe-Schriemer etal. in Sequencing an "Unknown" Peptide by Time-of-Flight Secondary IonMass Spectrometry (Int. J. Mass Spectrom. Ion Phys., 111, 301-315(1991)), the parent ions subjected to TOF-SIMS necessarily break down tothe various fragment ions, the masses of which can be compared andanalyzed based on existing mass data to determine the structure of theselected peptide. This procedure is effective to the extent that theselected molecular species is one of the possible peptides of thecombinatorial library as determined by the construction of the library.This procedure is also limited by the resolving power of TOF-SIMS todistinguish such fragmentions (TOF-SIMS mass accuracy is currently onthe order of ±0.01 amu, according to Winograd, supra).

Alternatively, an isotope indexing scheme can be used to differentiatebetween molecular species that otherwise have the same mass. Forexample, to differentiate between phenylalanine-glycine-leucine andglycine-leucine-phenylalanine, one can either examine the fragmentationpattern in the SIMS spectrum or synthesize one of the peptides usingleucine having ¹⁵ N, an isotope that is readily distinguished inTOF-SIMS as its atomic mass is increased by one unit. Distinguishingbetween a leucine and an isoleucine residue, which are isomers,necessarily would require such an alternate method. Similarly, one coulduse differentially L and D amino acids, using methods well known in theart.

In particular, the present invention relates to a method of identifyingsmall molecules of a combinatorial library comprising (a) forming aplurality of complexes of solid substrates and small molecules, each ofwhich comprises one substrate, or portion thereof, and one of said smallmolecules of said library; and (b) determining the molecular weight of aselected small molecule by means of secondary ion mass spectrometry.Preferably, the secondary ion mass spectrometry that is utilized in thecontext of the present invention is TOF-SIMS, as noted above andexemplified below. The small molecules of such a combinatorial libraryare selected from at least one of the group consisting of amino acids,peptides, oligonucleotides, and heterocyclic compounds. The presentinventive method is applicable to small molecules comprising amino acidsthat are naturally occurring or synthetic. A preferred combinatoriallibrary has small molecules that are peptides or heterocyclic compounds;a more preferred combinatorial library has small molecules that arepeptides.

Suitable peptides comprise as few as two amino acids to as many as about30; preferably, suitable peptides comprise from about two amino acids toabout fifteen; most preferably, suitable peptides comprise from abouttwo amino acids to about ten. Any amino acid may be incorporated intopeptides screened and identified using the present invention, includingany combination of the naturally occurring proteinogenic amino acids aswell as amino acids not naturally occurring in proteins such as, but notlimited to, dextrorotatory forms of the known amino acids, for example.

Suitable oligonucleotides consist of as few as two nucleotides to asmany as about 50; preferably, suitable oligonucleotides consist of fromabout five nucleotides to about 30; most preferably, suitableoligonucleotides consist of from about five oligonucleotides to about15. Any nucleotide may be incorporated into an oligonucleotide screenedand identified using the present invention, including any combination ofthe naturally occurring deoxyribonucleotides and ribonucleotides as wellas those not naturally occurring in biological systems, such as, but notlimited to, H-phosphonate derivatives,N-blocked-5'-O-DMT-deoxynucleoside3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites,N-blocked-5'-O-DMT-deoxynucleoside3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites,N-blocked-5'-O-DMT-deoxynucleoside 3'-(methyl-N,N-diisopropyl)phosphoramidites, N-blocked-5'-O-DMT-deoxynucleoside 3'-(2-chlorophenyl)phosphates, N-blocked-5'-O-DMT-deoxynucleoside 3'-(2-chlorophenyl2-cyanoethyl) phosphate, all of which are nucleoside derivatives used inoligonucleotide synthesis.

Suitable heterocyclic compounds consist of, at minimum, a single fourmembered ring to as much as a multiple of four membered or greatermembered rings coupled by carbon chains of 1 to about 20 atoms inlength, such chains being saturated or not. Preferably, suitableheterocyclic compounds include a single four- to seven-membered ring, aswell as, but not limited to varying combinations of 5, 6, or 7 memberedrings having varying numbers of N, S, or O atoms. More preferably,suitable heterocyclic compounds include benzodiazepine and derivativesthereof (as, for example, disclosed in Bunin et al., J. Am. Chem. Soc.,114, 10997-10998 (1992)), penicillins, cephalosporins, and folatederivatives. Most preferred, suitable heterocyclic compounds includebenzodiazepine and derivatives thereof, and angiotensin II receptorantagonists. For example, one angiotensin II receptor antagonist thatwas developed to block the renin-angiotensin system for the treatment ofheart failure and possibly chronic renal failure (see, Weinstock et al.,J. Med. Chem., 34, 1514 (1991); Keenan et al., J. Med. Chem., 36, 1880(1993)) can be identified in a mixture of other heterocyclic compoundsusing the present invention. The formula of the aforementionedangiotensin II receptor antagonist, ethyl 2-(2'-thiophenylmethyl)-3-5'-{(1'-p-carboxyphenylmethyl)-2'-n-butyl}-imidazolyl!-propenoate,covalently linked to polystyrene beads through various linking moietiesis set forth in FIG. 7. The present invention may be applied to theidentification of derivatives of such compunds as benzodiazepine and thenoted angiotensin II receptor antagonist.

Mixed libraries of small molecules comprising amino acids, peptides,oligonucleotides, and heterocyclic compounds may be prepared byfollowing standard methods known to one of ordinary skill in the art. Anoligonucleotide can be, for instance, linked to a peptide through the5'-hydroxyl of the oligonucleotide. The peptide end can be modified toinclude a carboxyl group. A process of esterification of the carboxylgroup with the 5'-hydroxyl of the oligonucleotide is used to produce amixed library containing peptide-oligonucleotide species. Brenner etal., (Proc. Nat'l Acad. Sci. USA, 89, 5381-5383 (1992) also describes amethod of preparation of mixed libraries having nucleotides andpeptides. A mixed library comprising a heterocyclic compound and apeptide is also prepared by the reaction of suitable functional groupspresent on the heterocyclic compound. For instance, the carboxyl groupon a heterocyclic compound is reacted with the amino group on thepeptide to provide an amide linkage.

The small molecules of the combinatorial library preferably are linkedcovalently to the substrate, using methods well known in the art. Apreferred covalent linkage between the small molecule and the substratehas the characteristic of being able to break in response to externalchanges at levels that do not modify substantially the structure of thesmall molecules of the combinatorial library. Such a covalent linkagemay be effected, for example, by means of a suitable linking moiety thatcouples both to the small molecule and the substrate or a substratehaving suitable reactive groups coupled thereto. In essence, a suitablecovalent linkage will break conditionally. When a linking moiety or asubstrate-bound reactive group is used, the covalent bonds between thesmall molecule and the substrate will break at one or more of itsinternal covalent bonds or a bond that it forms with either thesubstrate or the small molecule or both, thereby destroying any covalentlinkage between the small molecule and the substrate. At least anappreciable proportion of the population of small molecules will befully free of the covalent linkage, however, some or even a majority ofthe small molecules may remain attached covalently. The proportion ofsmall molecules whose covalent linkage to the substrate are broken,however, may remain associated with the substrate by weaker molecularinteractions, such as, but not limited to physical adsorption,hydrophobic interactions, and van der Waals forces. Suitable conditionchanges that may be used to effect the bond break or breaks of thecovalent linkage include effective levels of temperature,electromagnetic radiation, sound or acidity at a level that leaves thesmall molecules of the library intact but still in association with thesubstrate via some combination of the aforementioned or other weakmolecular interactions.

Suitable linking moieties are those that comprise a reactive functionalgroup selected from the group consisting of alcohol, amino, carboxyl,acetal, thioacetal, and aminoalkyl, aralkyl, amino aralkyl, andhaloalkyl, and a nitroaromatic group having a benzylic hydrogen ortho tothe nitro group, such as o-nitrobenzyl derivatives, and benzylsulfonylderivatives; and are cleavable by suitable vapor or photochemical means.Preferably, the linking moiety comprises at least one reactive groupthat is selected from the group consisting of hydroxyl, amino, carboxyl,acetal, thioacetal, C₁ -C₁₀ alkylamino, C₁ -C₁₀ aralkylamino, and C₁-C₁₀ haloalkyl, and an ortho-nitrobenzylic group having a benzylichydrogen. Photoremovable groups are discussed in U.S. Pat. No. 5,143,854to Pirrung et al., for example.

In particular, suitable linking moieties include p-alkoxybenzyl alcohol(used in the Wang resin),F-moc-2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine,F-moc-4-methoxy-4'-(gamma-carboxypropyloxy)-benzhydrylamine,4-hydroxymethyl-phenoxy-acetic acid, aminomethyl (used in the PAMresin), benzhydrylamine, Cl--CH₂ -Ph-(used in Merrifield resin),benzylacetal (used in the Acetal resin), benzylthioacetal (used in theThioacetal resin), and 2-methoxy-4-alkoxybenzyl alcohol (used in Sasrin®resin). Preferred linking moieties includeF-moc-2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine,F-moc-methoxy-4'(gamma-carboxypropyloxy)benzhydrylamine, p-alkoxybenzylalcohol, benzhydrylamine, Cl--CH₂ -Ph, 2-methoxy-4-alkoxy benzylalcohol, 6-nitroveratryloxy carbonyl, 2-nitrobenzyloxycarbonyl, andα,α-dimethyl-dimethoxybenzyloxycarbonyl, more preferred linking moietiesinclude 2-methoxy-4-alkoxybenzyl alcohol. It is appreciated thatdifferent linker chemistry may enhance the molecular ion signal ofcovalently attached species.

The covalent linkage between the substrate and the small molecule mayalso be mediated by the reactive group or groups attached to thesubstrate. For example, as recited above, the polystyrene-derivativebead known as Sasrin® (Bachem Biosciences) has a reactive group(2-methoxy-4-alkoxy benzyl alcohol) that covalently couples tocarboxylic acid groups found on all peptides. The covalent bond formedby the coupling of these two groups is acid labile. Accordingly, theexposure of TFA vapor to a small molecule covalently bound to a Sasrin®bead results in the breaking of certain covalent bonds associated withthe Sasrin® bead, i.e., the linking moiety, thereby releasing an intactmolecular species.

TFA is used preferably as a dilute solution in a suitable organicsolvent. The concentration of TFA is preferably kept in the range ofabout 0.5% to about 2% by weight, and more preferably from about 0.75%to about 1.5%, and most preferably from about 0.9% to about 1.1% byweight of the solution. The TFA is applied in concert with a means forswelling the polystyrene beads, such as, for example, dissolving the TFAin an organic solvent. Preferred organic solvents include halogenatedlower aliphatic hydrocarbons having 1-3 carbon atoms, includingmethylene chloride, chloroform, dichloroethane,1,1,2,2-tetrachloroethane, trichloroethylene, tetrachloroethylene, andthe like, with methylene chloride being more preferred.

The substrate upon or with which the small molecules of thecombinatorial library are synthesized and/or associated may be anysuitable substrate, including, but not limited to, resin, polystyrene,Sasrin®, Wang resin, Pam resin, and Merrifield resin, further includingsuitable combinations thereof. Such resins are commercially availablefrom Bachem Bioscience Inc., for example. The substrate used in thepresent invention may be formed into any suitable shape, including, butnot limited to, spheres, cubes, rectangular prisms, pyramids, cones,ovoids, sheets, and cylinders. Particularly when the substrate is usedin the form of a sheet, such as when placed on the surface of a glassmicroscope slide, defined portions of the sheet may be apportioned fordifferent small molecules of a combinatorial library, as disclosed inFodor et al., supra. Preferably, the substrate as used in the presentinvention is formed into small particles that occupy no more than nineten thousandths of a cubic millimeter, such as a sphere having adiameter of 120 microns, each of which has associated thereto a singlesmall molecule structure. More preferred, the substrate used in thepresent invention is a bead or sphere having a diameter that is fromabout 10 microns to about 120 microns. Most preferred, the substrateused in the present invention is a bead or sphere having a diameter thatis from about 20 microns to about 80 microns.

The present invention also relates to the linking moiety per se. Thecharacteristics and examples of the linking moiety are the same asdiscussed above relative to the method for identifying individual smallmolecules.

The following examples further illustrate the present invention and, ofcourse, should not be construed in any way as limiting its scope.

EXAMPLE 1

This example illustrates the use of TOF-SIMS for the identification ofthe molecular weights of combinatorial library constituent moleculesbound to the polystyrene bead surfaces. TOF-SIMS is an instrument thatis well known in the art and available from various commercial sources.Accordingly, an artisan may use any such TOF-SIMS in accordance with thespecific machine's operating instructions. What follows is a descriptionof the use of one TOF-SIMS instrument manufactured by Kratos, Inc.(Ramsey, N.J.), which was used in the context of the present invention.

A schematic diagram of the apparatus is shown in FIG. 1. An ion gun 100is used to generate a beam of primary ions directed at a bead 101 coatedwith a monolayer of the sample. The ion gun 100 is illustrative of theliquid metal type (LMIG), and provides a source of Ga⁺ ions having anenergy of 25 keV. The dosage of these ions is limited to stay within thestatic limit by limiting sample exposure to 200,000 pulses of 500 pAcurrent and 20 ns duration per pulse. This exposure corresponds to 10⁷Ga⁺ ions focused into a circular area of 40 μm diameter (the diameter ofthe bead) or 8×10¹¹ Ga⁺ ions/cm². A 20 ns primary ion pulse yields amass resolution of ˜1500 at m/z 100. Pulsing of the beam is achieved byrapid electrical deflection of the beam through an aperture for thedesired pulse duration. The ion beam is focused to a spot size ofapproximately 150 nm on the surface of the bead 101 through focusingoptics denoted generally by reference numeral 102. Since a plurality ofbeads are held on a single copper grid, the ion gun beam may be rastoredacross the surface, with spectra being taken at each pixel to determinethe surface constituents there.

Bombardment of a bead 101 by the ion beam causes the liberation ofsecondary ions from the surface. Secondary ions liberated from thesurface of the bead 101 by the incident ion pulse are then acceleratedto a uniform energy and are focused by an extraction lens 105. This lensis a combination of a flat extraction plate and an enzel lens. As willbe discussed in greater detail below, a constant voltage is maintainedbetween the copper grid 106 to which the bead is attached and theextraction lens 105. Preferably, the distance from the grid 106 to theextraction lens 105 is about 3 mm. Once through the extraction lens 105,the uniform energy ions travel along the linear path designatedgenerally by reference numeral 110. Focusing optics, preferably in theform of a reflectron 120, are placed at the end of the path 110. Thesefocusing optics correct for angular distribution of the secondary ions,as described in Cotter, Biomed. Environ. Mass Spec., 18, 513-532 (1989).The focused secondary ions are then detected by a channelplate detector130 located at the end of the TOF analyzer defined by path 110 andreflectron 120. Preferably, the length of the TOF analyzer is about 2 m.The channelplate detector 130 is connected to a computer 140, whichperforms processing required for spectrum analysis. Further electronics,not shown, are used for synchronizing the system so that the timebetween generation of secondary ions and their arrival at thechannelplate detector 130 is accurately measured.

To accelerate the secondary ions to a uniform energy, a constant voltageis maintained between the substrate 106 and the extraction lens 105.This voltage is preferably 7200 volts, with the copper grid being heldat +2.5 kV, and the extraction lens being held at -4.7 kV (forpositively charged secondary ions). The polarity and magnitude of thesesignals may be changed to allow for detection of negatively chargedspecies. Two mechanisms present in this configuration can lead to highersignals at the leading edge of a given bead. Because the ion currentdensities generated by the ion gun 100 are quite large, some charging ofthe sample occurs during bombardment. Further, because the bead has aphysical dimension (illustratively a 40 micron diameter) in the 3 mmextraction gap, a voltage gradient on the order of 150 V across the beadmay be present. The size of this gradient is affected by the size andshape of the bead and the angle of incidence of the Ga⁺ ion. Tocompensate for charging of the bead, the sample may be floodedperiodically with low energy electrons, such as 50 nA/cm² of 30 eVelectrons for 50 μs between each Ga⁺ ion pulse, to eliminate chargingartifacts.

EXAMPLE 2

This example illustrates the TOF-SIMS spectrum of a 40 micronpolystyrene bead coated with an approximately one molecular layer ofphenylalanine.

Standard 40 micron diameter polystyrene beads (Bachem Bioscience) weretreated with a solution of phenylalanine to cover the beads with amonolayer of the amino acid by physical adsorption, as follows:Polystyrene beads were immersed in a 10⁻⁴ M methanol solution ofphenylalanine, removed after several minutes, allowed to air dry, andthen placed on a copper grid for analysis. For these measurements, thedose of incident 25 keV Ga⁺ ions was controlled by limiting sampleexposure to 200,000 pulses of 500 pA current and 20 ns duration perpulse. This exposure corresponds to 10⁷ Ga⁺ ions focused into a circulararea of 40 pm diameter or 8×10¹¹ Ga⁺ ions/cm². A 20 ns primary ion pulseyields a mass resolution of ˜1500 at m/z 100. The low dose of primaryions ensures that sample damage does not alter the chemical nature ofthe target surface, as noted by Benninghoven and Sichterman (Anal.Chem., 50, 1180 (1978)).

As shown in FIG. 2A, the resultant TOF-SIMS spectrum exhibits largepeaks at m/z 120 (M--CO₂ H)⁺, 166 (M+H)⁺, 188 (M+Na)⁺, and 210(M+H+Na₂)⁺. Other peaks characteristic of bulk polystyrene (labeled "PS"at m/z 91 (C₇ H₇), 103, 105, 115, 117, 127, 128, 129, 141, 152, 165,178, 190, and 193; see Leggett et al., J. Chem. Soc. Faraday Trans., 88,297 (1992)), sodium (at m/z 23), also assignable.

Although sensitivity of the TOF-SIMS technique varies depending on themolecular character of the sample being tested, it is noteworthy thatfor phenylalanine adsorbed on a polystyrene bead, the detection limitwas approximately 500 attomoles on the bead surface.

Considering the capability of a 40 micron sphere to have adsorbed to itat least 50 femtomoles, i.e., at least 100 times above the detectionlimit, the TOF-SIMS technique was shown hereby to have the requisitesensitivity for analyzing combinatorial libraries according to thepresent invention.

Thus, this example illustrates the capability of TOF-SIMS to analyzesmall quantities of amino acids adsorbed on beads.

EXAMPLE 3

This example illustrates the TOF-SIMS spectrum of a 40 micronpolystyrene bead coated with an approximately one molecular layer of thetripeptide, valine-tyrosine-valine (V-Y-V).

Standard 40 micron diameter polystyrene beads (Bachem Bioscience Inc.)were treated with a solution of V-Y-V to cover the beads with amonolayer of the tripeptide by physisorption and then placed on a coppergrid, as described in Example 2. For the TOF-SIMS assay, the pulsed Ga⁺ion beam was rastored across the 100 micron field, during which time aTOF-SIMS spectrum was recorded (FIG. 3) for each -1 square micron pixel.An image was rendered by mapping the intensity of (M+H), (M+Na)⁺, and(M+H+Na₂)⁺ ions at m/z 380, 402 and 424, respectively. For V-Y-V, theintensity was generally 0-4 counts per pixel. In spite of theserelatively low count rates, a clear image of the coated bead wasdiscerned easily in a photograph of the noted intensity levels recordedand digitized in each pixel.

A number of points important in the interpretation of results derivedfrom the inventive method can be made with reference to the V-Y-Vanalysis. First, although the copper grid is electrically conductive,the polystyrene bead itself is an electrical insulator subject tocharging. Normally, in static TOF-SIMS experiments, charging is not asignificant problem, due to the small number of incident ions needed torecord a spectrum. For imaging of small areas, as done to generate theimage just mentioned, however, ion current densities are much higher,therefore some type of charge compensation is essential. In theexperiments accomplished in the course of elucidating the presentinvention, the sample was flooded with 50 nA/cm² of 30 eV electrons for50 microseconds between each Ga⁺ ion pulse to eliminate chargingartifacts, after the methods disclosed in Gardella and Hercules (Anal.Chem., 52, 226 (1980)) and Briggs and Wooton (Surf. and Int. Anal., 4,109 (1982)).

Second, the influence of the shape of the particle on which themolecular species of the library are attached and the angle of incidenceof the Ga⁺ ion stream have an impact on results. In the configurationused in the context of the present inventive method, the Ga⁺ beam wasincident at 45° from the surface normal to generate the data displayedin FIG. 3. For example, a polystyrene sphere of ˜60 microns in diameter,placed in a 3 mm extraction gap, will have a field of 7200 volts appliedacross it. Accordingly, in addition to problems dictated by themorphology of the bead, there is a 150 volt field gradient across thebead. Both of these effects tend to produce higher signals near theleading edge of the bead, as is visualized in the digitized images shownin FIG. 4, for example.

Third, each of the TOF-SIMS assays reported herein was completed in lessthan 4 minutes. The analysis time is determined by the flux of incidentions and the time required to reach the damage threshold. For smallbeads and/or higher current sources, the analysis time could be reducedsignificantly by about an order of magnitude.

Similar results to those shown in FIG. 3 have been obtained usingglycine-proline-glycine-glycine, as well as a variety of other smallpeptides. The technique for larger peptides, such as bradykinin, forexample, having 11 amino acid residues, provided a recognizable TOF-SIMSspectrum when the 11-mer was adsorbed onto a polystyrene film (seeSteffens et al., supra). Because combinatorial libraries of peptides onpolystyrene beads generally consist of linear chains of only three tosix amino acids, the range imaged by TOF-SIMS is certainly sufficient todetermine the parent molecular ion of the adsorbed peptides of suchlibraries.

Accordingly, using the tripeptide V-Y-V, this example provideselucidation of important parameters in the direct imaging of acombinatorial library of peptides adsorbed onto polystyrene beads. Onemust be cognizant of the charging capacity of the substrate to which themolecular species of a library are adsorbed because of the substrates'capacity to increase the ion current density. Additionally, the shape ofthe substrate used and the angle of incidence of the Ga⁺ ion can tend toproduce artificially higher signals, and therefore must be compensatedfor using methods well known in the art. Finally, the time per TOF-SIMSwas only four minutes, and could be reduced significantly, which is oneof the surprising improvements that the present invention provides tothe field of combinatorial library screening and analysis.

EXAMPLE 4

This example illustrates the determination of the molecular weight of apeptide at a particular address, using the TOF-SIMS assay as describedin Example 2 and a novel method to reversibly yet covalently link smallmolecules of a combinatorial library to a substrate.

Combinatorial libraries constructed on polyester beads are necessarilybound covalently thereto at least during the construction reactions. Forthe determination of the molecular weight of a small molecule of such alibrary located at a particular address, i.e., at a particular site on agrid, it is necessary to break the covalent linkage in order to desorbintact molecules. For the purpose of testing requirements of anaddress-based determination, phenylalanine was adsorbed onto Sasrin®polystyrene beads and linked covalently thereon by means of reactivegroups attached to the Sasrin® beads, using the methods of Mergler etal. I (Tet. Lett., 29, 4005 (1988)) and Mergler et al. II (Tet. Lett.,29, 4009 (1988)).

The formation of the covalent bond between phenylalanine and the Sasrin®bead dramatically reduced the yield of molecular ions in the SIMSspectrum. This effect is shown in FIG. 2B where the yield of (M+H)⁺ atm/z 166 is no longer visible, although strong fragment ions are found atm/z 120. Accordingly, the parent compound, phenylalanine, could not beidentified. When larger molecules were tested in otherwise identicalexperiments, parental molecular ions were not observed and the spectrawere found to consist mainly of intense fragment ions from protectinggroups and monomers, such as amino acids. For example, with thepentapeptide, leucine-serine-arginine-isoleucine-valine, the expectedparental molecular ion at 587 m/z was not observed nor were any of thecationized species, although several fragment ions typical of each ofthe monomer units were found in the low mass range. See Mantus et al.,Anal. Chem., 65, 1431 (1993). Hence, the TOF-SIMS analysis of smallmolecules covalently bound to polyester beads was determined to beineffective unless the covalent bond is broken prior to the TOF-SIMSanalysis.

A protocol for clipping the covalent bond or bonds that bind a smallmolecule of a library to a suitable substrate, while leaving the smallmolecule resting in place on the substrate, was developed usingphenylalanine attached to a Sasrin® polystyrene bead to test theprotocol. Beads with covalently attached amino acids were thentransferred to a copper grid. The copper grid was used as a support andmarkings on the grid were used to locate specific beads, i.e.,addresses.

It was discovered that Sasrin® polystyrene beads form acid sensitivecovalent bonds with peptides, for example. The beads having smallmolecules covalently attached thereto were placed in a chamber saturatedwith trifluoracetic acid (TFA) and methylene chloride (CH₂ Cl₂) vaporsfrom a 2% TFA in CH₂ Cl₂ solution. A three minute exposure wassufficient to cleave the amino acid from the bead. The progress of thereaction was monitored by observing a color change from off-white topurple on the beads themselves. Once the cleavage reaction was complete,the beads and copper grid were inserted directly into the TOF-SIMSinstrument for analysis.

The mass spectra of the beads subjected to the vapor phase clippingexhibited a strong signal for each corresponding parent ion. The SIMSspectrum of the clipped phenylalanine is shown in FIG. 2C, while thecorresponding image of m/z 166 is shown in FIG. 4. An importantobservation derived from FIGS. 4A and 4B, which are TOF-SIMS imagesdirected at the identical address, but using different filters, is thatthe peptide is confined to the bead. This is evident because its signal(shown in FIG. 4A) is not found from the surrounding copper grid (shownin FIG. 4B). Therefore, the phenylalanine after breaking its covalentlinkage to the polyester bead remained associated with the bead, due tophysical adsorption or other weak molecular effects. Moreover, thesignals observed for phenylalanine at m/z-(M+H)⁺ in FIG. 2C are moreintense than the same signal for phenylalanine when compared to thesignals when it was simply physically adsorbed to a bead (FIG. 2A),perhaps due to the better uniformity of coverage on the bead resultingfrom the covalent bond formation. Therefore, greater sensitivity resultsfrom analyzing molecular species clipped from the beads than fromanalyzing those prepared by other methods, such as by physisorptionalone.

The technique was further tested by imaging a mixture of phenylalanineand leucine coated beads, using the same procedure as above. The beadswere placed on a copper grid and cleaved with TFA as described above.The image is shown in FIG. 5, which is a field of view of 120 microns.Even though the beads were very close to each other there was nosignificant cross contamination, as seen by comparing FIG. 5A with FIG.5B, wherein the (M+H)⁺ ion intensity for leucine is shown in FIG. 5A andthe (M+H)⁺ ion intensity for phenylalanine is shown in FIG. 5B. As inFIG. 4, the images depicted in FIG. 5 are at the identical address,using different filters.

This example illustrates a method for further increasing the sensitivityof the TOF-SIMS molecular weight assay and, more significantly,illustrating a method for the determination of the molecular speciescontained in a library found at particular locations on a grid of beadscontaining different molecular species.

EXAMPLE 5

This example illustrates the TOF-SIMS assay as applied to theidentification of a tripeptide covalently bound to a bead.

The tripeptide Val-Tyr-Val was covalently attached through an acidsensitive linker to the bead, according to the method described inExample 4. The bead was subjected to clipping by the vapor phase methodand subjected to characterization by TOF-SIMS, as described in Example2. The mass spectrum displayed in FIG. 6 (lower panel) shows ions at m/z380 (M+H), 281, and 263. The assignment of these peaks is shown in thefigure, which are the whole tripeptide, a Val-Tyr dipeptide fragment,and a Tyr-Val dipeptide fragment, respectively. In the low mass range(FIG. 6, upper panel), intense peaks were found at m/z 72 (Val-Co₂ H)and 136 (Tyr-Co₂ --H). Using the method of Biemann et al., MassSpectrom. Rev., 6, 1 (1987), analysis of the TOF-SIMS spectrum offragment sequence ions provided not only the composition, but theVal-Tyr-Val sequence through the above fragmentation pattern.

Accordingly, this method provided a determination of the mass of theparent ion and thereby demonstrates a method to identify directly thosemembers of a library with a given molecular weight, as illustratedherein.

EXAMPLE 6

This example illustrates the use of the electrospray mass spectrometryto the identification of a heterocyclic small molecule covalently boundto the Sasrin® bead for comparison to the instant invention. The datadisclosed in this example and Example 7 were provided by S. A. Carr, M.E. Hemling, G. D. Roberts, and J. Weinstock of the Chemical andBiological Research Division of SmithKline Beecham Pharmaceuticals, Kingof Prussia, Pa.

A bead having attached thereto angiotensin II receptor antagonist (ethyl2-(2'-thiophenylmethyl)-3-5'-{(1'-p-carboxyphenylmethyl)-2'-n-butyl}-imidazolyl!-propenoate) wasisolated and transferred to a micro-Eppendorf tube. The analyte waspermitted to cleave for 15 min with 1% TFA in methylene chloride. Thesample was dried, and the compound was extracted/dissolved in 10 μlacetonitrile. One-tenth of the solution was introduced by flow injectionand analyzed by ESMS on a Perkin Elmer Sciex API-III triple quadrupoleanalyzer (Thornhill, Ontario). An intense signal corresponding to the(M+H)+ ion was readily detected at m/z 453.18 (theor. 453.18), as setforth in FIG. 8A. Strong signals from the bead and the linker were alsoobserved in the range m/z 500 to 600. An additional 10% of the solutionwas then analyzed by tandem MS on the same quadrupole instrument and theresult is set forth in FIG. 8B. The molecular ion cluster was selectedby Q1 of the triple quadrupole and collisionally activated with theargon in the collision cell, Q2. The product ions were detected in Q3. Alarge number of fragment ions were observed, all of which were readilyassigned to the structure of the angiotensin II receptor antagonist.

Thus, electrospray mass spectrometry could be used to determine themolecular weight of covalently bound heterocyclic compound.

EXAMPLE 7

This example illustrates the identification of the angiotensin IIreceptor antagonist covalently bound to the Sasrin® bead by using theMALDI method, presented here for comparative purposes.

The Sasrin® bead was placed on a stainless steel sample target andexposed for about 1 hr to TFA vapor in an enclosed chamber. A 0.5 μlalignment of a solution of dihydroxybenzoic (DHB) acid matrix in acetonewas placed on the bead, and allowed to dry in air. Analyses were carriedout using two types of Fisons VG MALDI mass spectrometers (Manchester,UK), both single-stage reflectron instruments using photon irradiationfrom a 337-nm pulsed nitrogen laser and 23-keV accelerating voltage. Forgenerating conventional MALDI spectra, a low performance TOFSpec withmaximum mass resolution in the reflecting mode of M/Δn 1200 (FWHM) wasused. A conventional MALDI mass spectrum was generated by 41 laser shotsthat were averaged to produce the spectrum set forth in FIG. 9A. Theinstrument was mass calibrated externally using the (M+H)+ peaks of DHBand gramicidin S. A significant signal is observed for the (M+H)+ at m/z453.15 (theor. 453.18). Fragments were not detected. Peaks below m/z 200are primarily due to the DHB matrix. DHB has been found to beparticularly useful for low M_(r) organic compounds because it gives avery low matrix background above m/z 200.

To overcome the fact that MALDI did not produce a significant fragmentspectrum for this compound, the post source decay (PSD) method (seee.g., Della-Negra et al., Anal. Chem., 57, 2035 (1985); Tang et al.,Anal. Chem., 60, 1791 (1988); Spengler et al., J. Phys. Chem., 96, 9678(1992); Kaufmann et al., J. Mass Spectrom., 131, 355 (1994)) was appliedto the angiotensin II receptor antagonist analysis using a VG TofSpec-SEinstrument. An approximately 10 Da window centered on the parent ion atm/z 453.2 was selected for product ion analysis using a Bradbury-Nielsonion gate. PSD mass spectra were acquired in seven consecutive,overlapping mass scale segments, each representing a ca. 30% mass changefrom the previous segment. The PSD segments were combined and externallymass calibrated against a PSD spectrum of renin substratetetradecapeptide by the data system to yield FIG. 9B. The spectrum wasreadily interpreted using well-developed rules for the interpretation offragment ions.

Thus Example 7 illustrates that the covalently bound heterocycliccompound can be clipped by TFA and its molecular weight determined byMALDI.

EXAMPLE 8

This example illustrates the identification of the angiotensin IIreceptor antagonist bound to the Sasrin bead by the TOF-SIMS method.

TOF-SIMS studies of the beads were carried out after exposure to thebeads of TFA vapor for a period of 2 hours in an enclosed chamber. Thebeads were supported on a small piece of silicon wafer. After the TFAvapor treatment, the wafer was transferred directly into the SIMSanalysis chamber of a Kratos Prism TOF-SIMS instrument, and the analysiswas carried out. A pulsed 25-keV primary ion beam (minimum beam diameter200 nm) irradiated the sample; the pulse width was 7 ns. A 2.5-keV stagevoltage was used to accelerate the ions into the analyzer, which is areflectron device capable of mass resolution better than m/Δm=10,000.The analytical data were output as either mass spectra or mass-resolvedimages.

FIG. 10 sets forth the positive ion spectrum obtained from a single beadwith a diameter of about 50 μm that had been obtained by bombarding with2.2×10⁷ Ga⁺ ions. Assuming 10¹⁴ molecules cm⁻², less than 0.5% of thesurface has experienced an ion impact. This is well within the staticlimit for SIMS. The (M+H)+ at m/z 453 is clearly evident together withthe significant fragment peaks at m/z 283, 135 and 97. The internalcalibration procedure using the H+ and the CH₃ + ions yield a m/z forthe (M+H)+ ion of 453.18 (theor. 453.18). The accuracy of thecalibration was checked by incorporating a CsI internal standard andusing the Cs+ at m/z 133. The fragment masses in FIG. 10 can bedetermined with similar accuracy, which also aids both in theirassignment and in their use along with the molecular mass in definingthe identity of the target compound.

The calculated and observed masses of fragment ions of the angiotensinII receptor antagonist detected in the illustrative examples 6-8 are setforth in Table 1 below. It is clear that the errors in the masses arenegligible and that the three mass spectrometric techniques describedabove are suitable for identifying polymer bound small molecules thatmay be present at picomole quantities. The TOF-SIMS method has evengreater sensitivity than MALDI as shown by the smaller magnitude of theerror in the experimental masses.

                                      TABLE 1                                     __________________________________________________________________________    Calculated and observed masses of fragment ions in FIGS. 8B, 9, and 10A                    Experimental masses (errors)                                     Composition                                                                          Calc. Mass                                                                          ESMS.sup.b                                                                            MALDI.sup.b                                                                           TOF-SIMS                                         __________________________________________________________________________    C.sub.5 H.sub.5 S                                                                    97.0112                                                                             96.96 (0.05)                                                                          97.0 (0.0)                                                                            97.015 (0.004)                                   C.sub.8 H.sub.7 O.sub.2                                                              135.0446                                                                            134.99 (0.05)                                                                         135.1 (0.1)                                                                           135.045 (0.000)                                  C.sub.11 H.sub.15 N.sub.2 O.sub.2                                                    207.1133                                                                            207.02 (0.09)                                                                         207.1 (0.0)                                                                           not obsd.                                        C.sub.11 H.sub.19 N.sub.2 O.sub.2                                                    235.1447                                                                            235.07 (0.07)                                                                         235.2 (0.1)                                                                           not obsd.                                        C.sub.15 H.sub.16 N.sub.2 O.sub.1 S                                                  272.0983                                                                            272.06 (0.04)                                                                         272.1 (0.0)                                                                           not obsd.                                        C.sub.17 H.sub.19 N.sub.2 O.sub.2                                                    283.1446                                                                            not obsd.                                                                             not obsd.                                                                             283.144 (0.001)                                  C.sub.19 H.sub.21 N.sub.2 O.sub.4                                                    341.1501                                                                            341.17 (0.02)                                                                         v. weak not obsd.                                        C.sub.21 H.sub.25 N.sub.2 O.sub.4                                                    369.1814                                                                            369.22 (0.04)                                                                         weak    not obsd.                                        C.sub.23 H.sub.23 N.sub.2 O.sub.3 S                                                  407.1429                                                                            407.21 (0.07)                                                                         407.1 (0.1)                                                                           not obsd.                                        C.sub.23 H.sub.25 N.sub.2 O.sub.4 S                                                  425.1535                                                                            425.22 (0.07)                                                                         425.0 (not                                                                            not obsd.                                                             measured)                                                C.sub.25 H.sub.29 N.sub.2 O.sub.4 S                                                  453.1848                                                                            453.18 (0.00).sup.a                                                                   453.15  453.183 (0.002)                                                       (0.03).sup.a                                             __________________________________________________________________________     .sup.a Values were obtained from normal spectra, not MS/MS or PSD             spectrum.                                                                     .sup.b ESMS and MALDI data were provided by S. A. Carr, M. E. Hemling, G.     D. Roberts, and J. Weinstock of the Chemical and Biological Research          Division of SmithKline Beecham Pharmaceuticals, King of Prussia,              Pennsylvania.                                                            

FIG. 11 sets forth two images obtained by collecting secondary ions asthe primary beam is rastored across the bead surface. The images showthe ion collection distribution for (A) the (M+H)+ ion and (B) the m/z135 ion. It is clear that the ions attributable to the target compoundare collected primarily from the bead and few ions are evident from thesilicon support. Thus, the images demonstrate that even after the TFAtreatment, the target compound remains substantially on the bead.

Thus, Example 8 illustrates that a heterocyclic compound covalentlybound to a substrate can be clipped by TFA and its molecular weightdetermined by TOF-SIMS.

EXAMPLE 9

This example sets forth the results of a TOF-SIMS assay of the presentinvention directed to the aforementioned angiotensin II receptorantagonist covalently attached to the Wang resin, the Acetal resin, orthe Thioacetal resin.

Angiotensin II receptor antagonist-substrate constructs were constructedusing the Wang resin, the Acetal resin, or the Thioacetal resin assubstrate. The antagonist-substrate covalent linkage was effected usinglinkers associated with the respective linkers, as shown in FIG. 7. Thecovalent linkages were clipped by exposure to TFA vapor. TOF-SIMS wasapplied to each of the above resin samples and the molecular weight ofthe antagonist was determined successfully.

The contents of each of the references identified herein are herebyincorporated by reference in their entirety.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A method of obtaining a molecular weight ofindividual small molecules of a combinatorial library comprising:(a)forming a plurality of complexes of solid substrates and said smallmolecules, each of said complexes comprising one substrate, or portionthereof, and at least one of said small molecules of said combinatoriallibrary, wherein said substrate and said small molecule are attached toone another by a covalent bond; (b) breaking said covalent bond suchthat said small molecule remains physically adsorbed to said substrate;and (c) determining the molecular weight by secondary ion massspectrometry of said small molecule adsorbed to said substrate.
 2. Themethod of claim 1, wherein said small molecules are selected from thegroup consisting of amino acids, peptides, oligonucleotides,heterocyclic compounds, and combinations thereof.
 3. The method of claim2, wherein said substrate comprises a polymeric resin having a linkingmoiety attached thereto.
 4. The method of claim 3, wherein saidpolymeric resin is a polystyrene resin having a linking moiety attachedthereto.
 5. The method of claim 4, wherein said linking moiety comprisesat least one reactive group that is selected from the group consistingof hydroxyl, amino, carboxyl, acetal, thioacetal, C₁ -C₁₀ alkylamino, C₁-C₁₀ aralkylamino, and C₁ -C₁₀ haloalkyl, and an o-nitrobenzylic grouphaving a benzylic hydrogen.
 6. The method of claim 5, wherein saidlinking moiety is selected from the group consisting ofF-moc-2,4-dimethoxy-4'-(carboxymethyloxy)-benzhydrylamine,F-moc-methoxy-4'(gamma-carboxypropyloxy)benzhydrylamine, p-alkoxybenzylalcohol, benzylacetal, benzylthioacetal, benzhydrylamine, Cl--CH₂ -Ph,2-methoxy-4-alkoxy benzyl alcohol, and o-nitrobenzyloxy carbonyl.
 7. Themethod of claim 6, wherein said linking moiety is selected from thegroup consisting of 2-methoxy-4-alkoxy benzyl alcohol, benzylacetal, andbenzylthioacetal.
 8. The method of claim 7, wherein said covalent bondis broken without substantial modification of said small molecule. 9.The method of claim 8, wherein said covalent bond is broken by using avapor comprising trifluoracetic acid.
 10. The method of claim 9, whereinsaid covalent bond is broken by using a mixture of trifluoracetic acidand methylene chloride vapors.
 11. The method of claim 10, wherein saidsubstrate is a bead.
 12. The method of claim 11, wherein said bead has adiameter of from about 10 microns to about 120 microns.
 13. The methodof claim 11, wherein said secondary ion mass spectrometry istime-of-flight secondary ion mass spectrometry.
 14. The method of claim13, wherein said method further comprises mapping of the spatialdistribution of said small molecules on said beads.
 15. The method ofclaim 12, wherein said small molecule is an amino acid or a peptide. 16.The method of claim 15, wherein said peptide comprises two to ten aminoacids.
 17. The method of claim 16, wherein said method further comprisesdetermination of the sequence of said peptide from the fragmentationpattern obtained in said time-of-flight secondary ion mass spectrometry.18. The method of claim 13, wherein said small molecule is aheterocyclic compound comprising four to seven membered rings having N,S, or O, and combinations thereof.
 19. The method of claim 1, whereinsaid substrate is a polystyrene bead having a reactive group, said smallmolecule is an amino acid, peptide, oligonucleotide, or a heterocycliccompound, or a combination thereof, said covalent bond is an acidsensitive ester bond, said covalent bond is broken by exposing saidcomplex placed on a grid to the vapors of trifluoracetic acid andmethylene chloride, and said secondary ion mass spectrometry istime-of-flight secondary ion mass spectrometry.